Gene deletions by ends-in targeting in Drosophila melanogaster

Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.     

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.Abbreviations ES embryonic stem - neo^r neomycin resistance gene - HSV herpes simplex virus - tk thymidine kinase gene - PCR polymerase chain reaction - LIF leukemia inhibitory factor - LTP long-term potentiation - Rb retinoblastoma gene product - CF cystic fibrosis     

Bacteriophage gene targeting vectors generated by transplacement

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.     

Genomic deletions of the Drosophila melanogaster Hsp70 genes

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned approximately 47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.     

How does the genetic assassin select its neuronal target?

Through many different routes of analysis, including human familial studies and animal models, we are identifying an increasing number of genes that are causative for human neurodegenerative disease and are now in a position for many such disorders to dissect the molecular pathology that gives rise to neuronal death. Yet a paradox remains: The majority of the genes identified cause neurodegeneration in specific neuronal subtypes, but the genes themselves are ubiquitously expressed. Furthermore, the different mutations in the same gene may cause quite different types of neurodegeneration. Something in our understanding of neurodegenerative disease is clearly missing, and we refer to this as the phenomenon of ??neuronal targeting.?? Here we discuss possible explanations for neuronal targeting, why specific neuronal subtypes are vulnerable to specific mutations in ubiquitously expressed genes.     

Design and packaging of adeno-associated virus gene targeting vectors

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.     

Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors

Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.     

AAV-mediated gene targeting methods for human cells

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ～10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.     

Oligonucleotide-directed mutagenesis and targeted gene correction: a mechanistic point of view

Within the last decade, a number of nucleic acid-based gene targeting strategies have been developed with the ultimate goal to cure human genetic disorders caused by mutations. Thus far, site-directed gene targeting is the only procedure that can make predefined alterations in the genome. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. In addition, targeted specific mutations can be made in the genome for functional analysis of proteins. Several approaches, including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small fragment homologous replacements, and triple-helix-forming oligonucleotides have been used for targeted modification of the genome. Due to the absence of standardized assays and mechanistic studies in the early developmental stages of oligonucleotide-directed gene alteration, it has been difficult to explain the large variations and discrepancies reported. Here, we evaluate the progress in the field, summarize the achievements in understanding the molecular mechanism, and outline the perspective for the future development. This review will emphasize the importance of reliable, sensitive and standardized assays to measure frequencies of gene repair and the use of these assays in mechanistic studies. Such studies have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The conventionally accepted but unproven dogmas of the mechanism of gene repair are challenged and alternative points of view are presented. Another important focus of this review is the development of general selection procedures that are required for practical application of this technology.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

The "image" of the regulatory gene in experiments with Drosophila

The mutants referred to as facultative dominant lethals were selected in the progeny of gamma-irradiated Drosophila males. The mutant males were viable and fertile, though their crosses with females of the yellow line yielded no daughters. The mutations obtained differed from the common mutations by (1) extremely varying penetrance of F1 hybrids from crosses with various lines; (2) the uncertain relationships between the mutant and normal alleles; (3) the different expression in somatic and germ cells; (4) the dependence of the expression on the sex of the parent carrying the donor mutations; (5) the mass morphosis formation and (6) the frequent reversal to the norm. These mutations are assigned to the regulatory group and their specific expression (see above) can be helpful in identifying regulatory gene mutations. We assume that the specific expression of the mutations studied is related to specific properties of the regulatory genes. These properties are as follows: (1) only one out of two homologous regulatory genes located on one homolog is in an active state, (2) in the haploid chromosome set the regulatory gene is represented by several alleles (cys-alleles); (3) only one allele ensures the regulatory gene activity.     

Genetic analysis of the ADGF multigene family by homologous recombination and gene conversion in Drosophila

Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.     

Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis

Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.     

Specific genetic modifications of domestic animals by gene targeting and animal cloning

The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.     

Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting

The creation of precise clinical mutations by gene targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.     

Proto-oncogenes in mammalian development.

The phenotypic analysis of mice carrying germline mutations in protooncogenes is beginning to provide convincing genetic evidence for the important role that these genes play in mammalian development and differentiation. Two approaches are being taken to elucidate the biological function of proto-oncogenes in vivo. The first involves the molecular analysis of existing mouse developmental mutants, while the second approach involves the generation of specific germline mutations by gene targeting using homologous recombination in embryonic stem cells. Several key points have already emerged from these genetic approaches. First, many proto-oncogenes are important to more than one cell lineage and function both during embryogenesis and in the adult. Second, the patterns of expression of these genes provide only a guide to their biological function. Third, mutant phenotypes are generally less severe than would be expected from their expression patterns, suggesting that there may be functional overlap between two or more members of a gene family.     

A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice.

Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics. The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance. Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells. The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts. By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements.